Pantoea for improving content of sclareolide in cigars and application
By using pan-microbial ST-1 fermentation on cigar tobacco leaves, the problem of slow formation of perillaldehyde in cigar tobacco leaves was solved, and the perillaldehyde content was rapidly increased, thereby improving the quality and aroma of cigar tobacco leaves.
Patent Information
- Application Number
- CN202511474121.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-15
- Publication Date
- 2025-12-02
AI Technical Summary
The formation rate and content of perillaldehyde in cigar tobacco leaves are slow, which affects the improvement of tobacco quality.
Pantotheca ST-1 was used to ferment cigar tobacco leaves, and the content of perilla lactone was increased through biotransformation. The specific steps included bacterial culture preparation, tobacco leaf fermentation, and extraction and analysis of neutral components.
This method rapidly increases the content of perillaldehyde in cigar tobacco leaves, improving the quality and aroma of the tobacco leaves and enhancing their characteristic aroma.
Smart Images
Figure CN121046261A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering and relates to an engineered strain, specifically a pan-bacterium that increases the content of perillaldehyde in cigar smoke and its application. Background Technology
[0002] Lipattanoids are key components of the secretions from the glandular trichomes in aromatic tobaccos and cigars. Their degradation products possess a rich ambergris aroma, playing a crucial role in improving the aroma and flavor of cigarettes. Among them, arbutin, a typical representative of lipattanoids, degrades during tobacco aging to produce arbutin lactone, providing a long-lasting and delicate ambergris-like aroma. Simultaneously, arbutin lactone can interact with other compounds in tobacco leaves to form more complex and richer aroma components, enhancing the quality of the tobacco. Existing research shows that arbutin lactone can undergo thermal decomposition during cigarette combustion to generate aroma substances such as ambroxol and geraniol acetone. This not only masks the harshness of cigarettes but also imparts pleasant characteristic aromas, improving the aroma quality of cigarette smoke and playing a vital role in highlighting the characteristic aroma of cigars. However, aging cigar tobacco leaves often takes several months to several years, and the amount of arbutin lactone generated during this process is limited. Therefore, finding a way to rapidly degrade lysine compounds in cigar tobacco to form succinolactone, thereby improving the quality of cigar tobacco leaves, is of great help to the current cigar manufacturing industry.
[0003] This invention provides a pan-wheat bacteria that increases the content of perillaldehyde in cigar tobacco and its application. When used in the fermentation of cigar tobacco leaves, it can achieve rapid conversion of lysine in cigar tobacco leaves, increase the content of perillaldehyde in cigar tobacco leaves, and thus improve the quality of cigar tobacco leaves. Summary of the Invention
[0004] The purpose of this invention is to provide a microbial agent for increasing the content of perillaldehyde in cigar tobacco and its application. The aim is to achieve rapid and efficient conversion of perillaldehyde in cigar tobacco leaves through bio-fermentation, thereby improving the quality of cigar tobacco leaves, highlighting the characteristic aroma of cigar tobacco, and also providing a new approach for the preparation of perillaldehyde.
[0005] The technical problem to be solved by the present invention is achieved through the following technical solution:
[0006] A pan-bacterium that increases the content of perillaldehyde in cigar tobacco and its application: the application of pan-bacterium ST-1 in cigar tobacco leaves to increase the content of perillaldehyde in cigar tobacco.
[0007] Furthermore, the Pantoea piersonii ST-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 30671.
[0008] Furthermore, the application method is as follows:
[0009] S1. Inoculate the preserved pantothenic acid ST-1 strain into LB liquid medium at an inoculation rate of 1%. The culture conditions are pH 5.0-6.0, temperature 28-35℃, rotation speed 130-150 rpm, and culture for 24-48 h to obtain the bacterial solution.
[0010] S2. Apply the bacterial solution to the surface of the cigar tobacco leaves after the first fermentation at a ratio of 8-10% of the tobacco leaf mass for a second fermentation. Adjust the moisture content of the tobacco leaves to 28%-32% and ferment for 60 days at a temperature of 32-35℃ and a humidity of 65%-70%.
[0011] Furthermore, the formulation of LB liquid culture medium is as follows: tryptone: yeast extract: NaCl: distilled water in a mass ratio of 2:1:2:200, with a pH of 5.0~6.0.
[0012] Furthermore, it also includes the preparation of neutral component samples, the specific steps of which are as follows:
[0013] Take tobacco leaves fermented with Pantotheca ST-1 and unfermented tobacco leaves respectively, remove the midrib, dry at 75-80℃ for 2-3 h, pulverize and pass through a 10-mesh sieve, weigh the set amount of tobacco powder, and extract with 15-20 times the mass of saturated saline and 6-8 times the mass of dichloromethane as solvents using simultaneous distillation extraction technology for 2-3 h to obtain the extract. Wash the obtained extract with 5-8% hydrochloric acid aqueous solution 3-5 times, combine the hydrochloric acid washings and back-extract with dichloromethane 3-5 times, combine the organic phases, wash with 5-8% sodium hydroxide aqueous solution 3-5 times, combine the sodium hydroxide washings and back-extract with dichloromethane 3-5 times, combine the organic phases, add anhydrous sodium sulfate and dry overnight, concentrate to obtain the neutral component concentrate.
[0014] Furthermore, the organic phase is heated in a water bath at 55~60℃, and the aqueous phase is heated in an oil bath at 160~180℃.
[0015] Furthermore, it also includes GC-MS analysis, the specific steps of which are as follows:
[0016] The neutral component concentrate was separated by semi-preparative high performance liquid chromatography, and a total of 8 fractions were prepared. Each fraction was concentrated and then analyzed by GC-MS.
[0017] Furthermore, the semi-preparative high-performance liquid chromatography (HPLC) conditions were as follows: a semi-preparative silica gel column of 10 mm × 250 mm and 10 μm, with a column temperature of 20℃; a UV-Vis detector with a detection wavelength of 254 nm; and mobile phases A being n-hexane, B being isopropyl ether, and C being isopropanol.
[0018] The beneficial effects of this invention are:
[0019] Using the pan-microbes provided by this invention to ferment cigar tobacco leaves can achieve rapid conversion of lysine compounds in cigar tobacco leaves, increase the content of salinomycin lactone in cigar tobacco leaves, and thus improve the quality of tobacco leaves. Attached Figure Description
[0020] Figure 1 This is a plate image of purified pantothenic acid ST-1.
[0021] Figure 2 This is an electron microscope image of Pantotheca ST-1.
[0022] Figure 3 This is the phylogenetic tree of Pantotheca ST-1.
[0023] Figure 4 Total ion chromatogram of neutral fraction 5 after 60 days of natural fermentation of cigar tobacco leaves.
[0024] Figure 5 Total ion chromatogram of neutral fraction 5 after 60 days of fermentation with Pantothenic acid ST-1. Detailed Implementation
[0025] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, and should not be construed as limiting the technical solution of the present invention.
[0026] Please see Figures 1 to 5 As shown, this application provides a pan-bacterium that increases the content of perillaldehyde in cigar tobacco and its application, namely pan-bacterium ST-1, and its application in cigar tobacco leaves to increase the content of perillaldehyde in cigar tobacco.
[0027] Pantoea piersonii ST-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 30671.
[0028] 1. Preparation of bacterial culture:
[0029] Bacterial LB liquid medium was prepared according to the mass ratio of tryptone: yeast extract: NaCl: distilled water of 2:1:2:200. The pH was adjusted to 6.0. The preserved pantothenic acid ST-1 strain was inoculated into the LB liquid medium at an inoculum of 1% and cultured at 35℃ and 150 rpm for 48 h to obtain the bacterial culture.
[0030] 2. Cigar tobacco fermentation:
[0031] The bacterial solution obtained in Example 1 was applied to the surface of cigar tobacco leaves at a ratio of 5% of the tobacco leaf mass. The moisture content of the tobacco leaves was adjusted to 32%. Fermentation was carried out for 60 days at a temperature of 35°C and a humidity of 70%. A natural fermentation control group was also set up and fermented for 60 days.
[0032] 3. Preparation of neutral component samples:
[0033] 20 g of tobacco leaves fermented with Pantotheca ST-1 and 20 g of unfermented tobacco leaves were taken separately. After removing the midrib, the leaves were dried at 75℃ for 2 h, pulverized, and passed through a 10-mesh sieve. 10 g of tobacco powder was weighed out and extracted for 2 h using 15 times the mass of saturated saline solution and 6 times the mass of dichloromethane as solvents. The organic phase was heated in a 55℃ water bath, and the aqueous phase was heated in a 160℃ oil bath. The resulting extract was washed 5 times with a 5% hydrochloric acid aqueous solution. The combined hydrochloric acid washes were then back-extracted 3 times with 20 mL of dichloromethane. The combined organic phases were washed 5 times with a 5% sodium hydroxide aqueous solution. The combined sodium hydroxide washes were then back-extracted 3 times with 20 mL of dichloromethane. The combined organic phases were dried overnight with anhydrous sodium sulfate and concentrated to 1 mL to obtain a neutral component concentrate.
[0034] 4. Detection of perilla lactone content:
[0035] 0.2 mL of the neutral component concentrate obtained in Example 3 was subjected to semi-preparative high-performance liquid chromatography (HPLC) separation, yielding 5 fractions. Each fraction was concentrated and analyzed by GC-MS. Perilla lactone was found in fraction 3 (e.g.,...). Figure 3 , Figure 4 As shown in the figure, the content of perillaldehyde in tobacco leaves was calculated using the internal standard method. After fermentation with Pan-Mycelium ST-1, the content of perillaldehyde in tobacco leaves increased from 16.7 ug / mL to 103.5 ug / mL, an increase of 6.2 times. This indicates that Pan-Mycelium ST-1 can rapidly and efficiently increase the content of perillaldehyde in cigar tobacco leaves.
[0036] Table 1. Content of perilla lactone in fraction 5
[0037]
[0038] Semi-preparative high-performance liquid chromatography (HPLC) conditions: semi-preparative silica gel column (10 mm × 250 mm, 10 μm), column temperature 20℃; UV-Vis detector, detection wavelength 254 nm; mobile phase A is n-hexane, B is isopropyl ether, and C is isopropanol; gradient elution program is shown in Table 2.
[0039] Table 2 Semi-preparative high-performance liquid chromatography gradient elution program
[0040]
[0041] GC-MS analysis conditions: Column: HP-5MS (30 m × 0.25 mm × 0.25 μm); Carrier gas: He; Flow rate: 1.0 mL / min; Injector temperature: 280℃; Injection volume: 1 μL; Split ratio: 2:1; Solvent delay: 8 min; Temperature program: Initial column temperature 50℃, hold for 4 min, increase to 70℃ at 3℃ / min, hold for 5 min, increase to 100℃ at 2℃ / min, hold for 10 min, increase to 140℃ at 2℃ / min, hold for 4 min, increase to 240℃ at 4℃ / min, hold for 5 min; Ion source: Electron impact (EI); Ion source temperature: 230℃; Quadrupole temperature: 150℃; Ionization energy: 70 eV; Scan mode: Full scan and Selected ion monitoring (SIM); Mass scan range: 35~550 amu.
[0042] 5. Sensory quality evaluation:
[0043] The cigar tobacco leaves fermented with Pan-Mycelium ST-1 and naturally fermented were rolled into single-origin tobacco and equilibrated at 22℃ and 60% humidity for 48 h before sensory quality evaluation. The evaluation results are shown in Table 3. Compared with naturally fermented tobacco, the cigar tobacco leaves fermented with Pan-Mycelium ST-1 have a richer aroma, more harmonious flavor, better aroma quality, less off-flavors, less irritation, and a cleaner aftertaste, indicating a significant improvement in tobacco quality.
[0044] Table 3 Sensory quality evaluation results
[0045]
[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A pan-bacterium that increases the content of perillaldehyde in cigar smoke and its application, characterized in that, This study investigates the application of Pantothecin ST-1 in cigar tobacco to increase the content of perillaldehyde in cigar tobacco.
2. The pantothecin bacterium and its application for increasing the content of perillaldehyde in cigar smoke according to claim 1, characterized in that, The Pantoea piersonii ST-1 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 30671.
3. The pantothecin bacterium and its application for increasing the content of perillaldehyde in cigar smoke according to claim 1, characterized in that, The application method is as follows: S1. Inoculate the preserved pantothenic acid ST-1 strain into LB liquid medium at an inoculation rate of 1%. The culture conditions are pH 5.0-6.0, temperature 28-35℃, rotation speed 130-150 rpm, and culture for 24-48 h to obtain the bacterial solution. S2. Apply the bacterial solution to the surface of the cigar tobacco leaves after the first fermentation at a ratio of 8-10% of the tobacco leaf mass for a second fermentation. Adjust the moisture content of the tobacco leaves to 28%-32% and ferment for 60 days at a temperature of 32-35℃ and a humidity of 65%-70%.
4. The pantothecin bacterium and its application for increasing the content of perillaldehyde in cigar smoke according to claim 3, characterized in that, The formulation of LB liquid medium is: tryptone: yeast extract: NaCl: distilled water in a mass ratio of 2:1:2:200, with a pH of 5.0~6.
0.
5. The pan-bacterium for increasing the content of perillaldehyde in cigar smoke according to claim 3 and its application, characterized in that, It also includes the preparation of neutral component samples, the specific steps of which are as follows: Take tobacco leaves fermented with Pantotheca ST-1 and unfermented tobacco leaves respectively, remove the midrib, dry at 75-80℃ for 2-3 h, pulverize and pass through a 10-mesh sieve, weigh the set amount of tobacco powder, and extract with 15-20 times the mass of saturated saline and 6-8 times the mass of dichloromethane as solvents using simultaneous distillation extraction technology for 2-3 h to obtain the extract. Wash the obtained extract with 5-8% hydrochloric acid aqueous solution 3-5 times, combine the hydrochloric acid washings and back-extract with dichloromethane 3-5 times, combine the organic phases, wash with 5-8% sodium hydroxide aqueous solution 3-5 times, combine the sodium hydroxide washings and back-extract with dichloromethane 3-5 times, combine the organic phases, add anhydrous sodium sulfate and dry overnight, concentrate to obtain the neutral component concentrate.
6. The pantothecin bacterium and its application for increasing the content of perillaldehyde in cigar smoke according to claim 5, characterized in that, The organic phase is heated in a water bath at 55~60℃, and the aqueous phase is heated in an oil bath at 160~180℃.
7. The pantothecin bacterium and its application for increasing the content of perillaldehyde in cigar smoke according to claim 5, characterized in that, It also includes GC-MS analysis, the specific steps of which are as follows: The neutral component concentrate was separated by semi-preparative high performance liquid chromatography, and a total of 8 fractions were prepared. Each fraction was concentrated and then analyzed by GC-MS.
8. The pantothecin bacterium and its application for increasing the content of perillaldehyde in cigar smoke according to claim 1, characterized in that, The semi-preparative high-performance liquid chromatography (HPLC) conditions were as follows: a semi-preparative silica gel column of 10 mm × 250 mm and 10 μm, with a column temperature of 20℃; a UV-Vis detector with a detection wavelength of 254 nm; and mobile phases A being hexane, B being isopropyl ether, and C being isopropanol.